DNA Microarray:
A Recombinant DNA Method
Basic Steps to Microarray:
Obtain cells with genes that are needed for analysis.
Isolate the mRNA using extraction buffer.
Remove the buffer with RNA.
The mRNA will be 5’-3’ with repeating AAA..end.
Convert each mRNA into colored cDNA.
Targets labeled with fluorophores.
Basic Steps to Microarray:
With mRNA degraded reverse cDNA created.
Mix colored samples of cDNA.
Microarray or DNA Chip:
Every spot on the chip represents a different coding sequence from different genes.
Each spot on the chip is made of a DNA probe that can pair with the cDNA that was created.
Basic Steps to Microarray:
Incubation with the mixed cDNA and the chip DNA will yield some pairing.
Wash off unbound cDNA to see what has bound to the microarray.
Visualizing Bound cDNA:
The slide with the microarray chip is placed inside a dark box where it is scanned with a high resolution laser that detects the bound fluorescent labels.
The information and images are then sent to the computer for analysis.
Analyzing the Data:
Creates a ratio image.
Green images signal expression in one condition.
Red images signal expression in one condition.
Yellow images signal expression in both conditions.
Capacities:
Not limited to human genetic material.
Can be used for all species.
Can display thousands of different genes.
Pending chip size.
100’s nucleotides (<1 cm^2)
100,000’s nucleotides (about 10 cm^2)
Allows the study of multiple genes at once.
Types of Microarray:
Pre-synthesized nucleic acids.
Bought cDNA.
Oligonucleotide synthesis in situ.
Creating cDNA in question.
Microarray Application:
Identifying gene’s and gene mutations of different types of diseases such as cancer.
Identifying the expression level, or quantity of genes (mutated or not).
Definitions:
Target - the nucleic acid (cDNA) sample who’s identity and quantity are being measured.
Fluorophore – usually green and red labels attached to the target to enable visualizing expression.
Microarray works as reverse hybridization method converting from mRNA to cDNA 3’-5’ with TTTT…end.
Probe – an attached nucleic acid with a known sequence (the DNA chip).
Resources:
Strachan, Tom; Read, Andrew. Human Molecular Genetics 3. pp 175-178. New York. Garland Publishing. 2004.
Campbell,A. Malcom. Microarray Animation. Davidson College. 2001. www.bio.davidson.edu/courses/genomics/chip/chip.html
http://cmgm.stanford.edu
http://www.gene-chips.com/